Anti ezh2

For preparation of soluble protein in cell lysates, cells were first washed twice with ice-cold PBS and sonicated in 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% (w/v), Nonidet P-40 with protease(s) and 25 mM NaF, 2 mM Na₃VO₄, 0.1 mM PMSF, and 20 μg/mL aprotinin. The soluble and insoluble fractions were prepared by centrifugation at 15,000 × g for 15 min at 4°C. The soluble proteins were separated by electrophoresis through SDS-polyacrylamide gels (6, 8, and 15% w/v acrylamide) and then electrophoretically transferred to a PVDF membrane. [Subsequently, each membrane was blocked with 5% (w/v) skim milk in Tris-buffered saline containing 0.1% (w/v), Tween-20 for 1 h at room temperature and then hybridized with the appropriate primary antibody (diluted in tris-buffered saline) with gentle agitation overnight at 4°C. After washing three times with this buffer, each membrane was incubated with an appropriate secondary antibody for 1 h at room temperature. Each immunopositive band was visualized with enhanced chemiluminescence detection (GE Healthcare). The following antibodies and chemicals were used: anti-EZH2 (1:1000; BD), anti-H3K27me3 (1:1000; Abcam), anti-histone H3 (1:1000; Santa Cruz Biotechnology), and anti-α-tubulin (1:5000; Sigma); MG-132 was from Millipore, and 3-deazaneplanocin A-HCl was from Cayman Chemical Company. Images were quantified with Image J software.

Lysates were prepared in RIPA buffer [16 (link)], and western blotting was performed as described [17 (link)]. The following antibodies were used for western blot analysis: anti-Ezh2 (BD Biosciences #612666), anti-Suz12 (Cell Signaling 3737S), anti-Eed (Millipore 05–1320), anti-H3K27me1 (Millipore 07–448), anti-H3K27me2 (Millipore 07–452), anti-H3K27me3 (Millipore 07–449), anti-Histone 3 (Millipore 07–690), anti-β-actin (Sigma A5441), anti-Gapdh (Sigma G8795), anti-ERα (Millipore 07–690), anti-Ezh1 (Millipore 07–690), anti-PARP (Cell Signaling #9542), anti-p16 (Santa Cruz M-156), and anti-p19 (Rockland Immunochemicals #200-501-891). Secondary antibodies included HRP-conjugated anti-rabbit and anti-mouse (Southern Biotech), both 1:10,000.

For total cell lysate extraction, cells were washed twice with ice-cold PBS and lysed in NETN buffer (20 mM Tris at pH 8.0, 150 mM NaCl, 1 mM EDTA at pH 8.0, 0.5% Nonidet P-40) with protease and phosphatase inhibitors (25 mM NaF, 2 mM Na3VO4, 0.1 mM PMSF, 20 μg/mL aprotinin) by sonication. Samples (30 μg) were run on 10% SDS-polyacrylamide gels, and the separated proteins were then blotted onto polyvinylidene difluoride (PVDF) membranes. The primary antibodies used were mouse monoclonal anti-EZH2 (BD Biosciences, 1:1000) and anti-β-actin (Sigma, St. Louis, MO, USA, 1:5000) and rabbit polyclonal anti-CXCR4 (Abcam, Cambridge, UK, 1:1000). The secondary antibodies used were a horseradish peroxidase-conjugated rat anti-mouse secondary antibody (Merck Millipore, Danvers, MA, USA, 1:5000) and an anti-rat secondary antibody (Merck Millipore, Danvers, MA, USA, 1:5000). Immunoreactive bands were visualized with the ECL Detection Reagent (GE, Piscataway, NJ, USA).

ChIP was performed as described previously [37 (link)]. Briefly, fresh liver tissue was cut into small pieces, then cross-linked with 1% formaldehyde, sonicated, pre-cleaned, and incubated with 5-10 μg of antibody per reaction. After being mixed with magnetic beads for 2 hours, complexes were washed with low and high salt buffers, and the DNA was extracted and precipitated. The enrichment of the DNA template was analyzed by conventional PCR using primers specific for each target gene promoter. The sequences of the primers were for p16-a, 5′-GCAACAGGGAATGGAACT-3′ and 5′-AGGTATCTGGGCAGAAGG-3′ (−1800bp to −1400bp upstream of the TSS), for p16-b, 5′-CAAAGTCACATACTAGAGGGAA-3′ and 5′-GGGTCTTATAGAGCGGATT-3′ (−1400bp to −1000bp), and for p16-c, 5′-CTTCCCGCTTTCTCAATCTCC-3′ and 5′-CCCGGCTCTTCCTCTTTCC-3′ (−1000bp to −600bp). Antibodies used in ChIP assay were anti-CUL4B (Sigma), anti-EZH2 (BD, Franklin Lakes, NJ, USA), anti-H3K27me3 (Millipore) and anti-H2AK119ub1 (CST).